Compounds and methods for inhibiting selectin-mediated function

ABSTRACT

Compounds and methods are provided for modulating in vitro and in vivo processes mediated by selectin binding. More specifically, selectin modulators and their use are described, wherein the selectin modulators that modulate (e.g., inhibit or enhance) a selectin-mediated function comprise a class of compounds termed BASAs (Benzyl Amino Sulfonic Acids, which include a portion or analogue thereof) linked to a carbohydrate or glycomimetic.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/411, 266 filed Apr. 26, 2006, now allowed; which application is acontinuation of U.S. patent application Ser. No. 10/440,476 filed May16, 2003, now issued as U.S. Pat. No. 7,060,685; which applicationclaims the benefit under 35 U.S.C. §119(e) of U.S. Provisional PatentApplication No. 60/381,214 filed May 16, 2002; which applications areincorporated herein by reference in their entireties.

BACKGROUND

1. Technical Field

The present invention relates generally to compounds, compositions andmethods for modulating processes mediated by selectin binding, and moreparticularly to selectin modulators and their use, wherein the selectinmodulators that modulate a selectin-mediated function comprise a classof compounds termed BASAs (Benzyl Amino Sulfonic Acids, which include aportion or analogue thereof) linked to a carbohydrate or glycomimetic.

2. Description of the Related Art

When a tissue is infected or damaged, the inflammatory process directsleukocytes and other immune system components to the site of infectionor injury. Within this process, leukocytes play an important role in theengulfment and digestion of microorganisms. Thus, the recruitment ofleukocytes to infected or damaged tissue is critical for mounting aneffective immune defense.

Selectins are a group of structurally similar cell surface receptorsthat are important for mediating leukocyte binding to endothelial cells.These proteins are type 1 membrane proteins and are composed of an aminoterminal lectin domain, an epidermal growth factor (EGF)-like domain, avariable number of complement receptor related repeats, a hydrophobicdomain spanning region and a cytoplasmic domain. The bindinginteractions appear to be mediated by contact of the lectin domain ofthe selectins and various carbohydrate ligands.

There are three known selectins: E-selectin, P-selectin and L-selectin.E-selectin is found on the surface of activated endothelial cells, whichline the interior wall of capillaries. E-selectin binds to thecarbohydrate sialyl-Lewis^(x) (SLe^(x)), which is presented as aglycoprotein or glycolipid on the surface of certain leukocytes(monocytes and neutrophils) and helps these cells adhere to capillarywalls in areas where surrounding tissue is infected or damaged; andE-selectin also binds to sialyl-Lewis^(a) (SLe^(a)), which is expressedon many tumor cells. P-selectin is expressed on inflamed endothelium andplatelets, and also recognizes SLe^(x) and SLe^(a), but also contains asecond site that interacts with sulfated tyrosine. The expression ofE-selectin and P-selectin is generally increased when the tissueadjacent to a capillary is infected or damaged. L-selectin is expressedon leukocytes. Selectin-mediated intercellular adhesion is an example ofa selectin-mediated function.

Modulators of selectin-mediated function include the PSGL-1 protein (andsmaller peptide fragments), fucoidan, glycyrrhizin (and derivatives),anti-selectin antibodies, sulfated lactose derivatives, and heparin. Allhave shown to be unsuitable for drug development due to insufficientactivity, toxicity, lack of specificity, poor ADME characteristicsand/or availability of material.

Although selectin-mediated cell adhesion is required for fightinginfection and destroying foreign material, there are situations in whichsuch cell adhesion is undesirable or excessive, resulting in tissuedamage instead of repair. For example, many pathologies (such asautoimmune and inflammatory diseases, shock and reperfusion injuries)involve abnormal adhesion of white blood cells. Such abnormal celladhesion may also play a role in transplant and graft rejection. Inaddition, some circulating cancer cells appear to take advantage of theinflammatory mechanism to bind to activated endothelium. In suchcircumstances, modulation of selectin-mediated intercellular adhesionmay be desirable.

Accordingly, there is a need in the art for identifying inhibitors ofselectin-mediated function, e.g., of selectin-dependent cell adhesion,and for the development of methods employing such compounds to inhibitconditions associated with excessive selectin activity. The presentinvention fulfills these needs and further provides other relatedadvantages.

BRIEF SUMMARY

Briefly stated, this invention provides compounds, compositions andmethods for modulating selectin-mediated processes. In one aspect of thepresent invention, the compounds that modulate (e.g., inhibit orenhance) a selectin-mediated function contain a BASA (i.e., a benzylamino sulfonic acid or portion or analogue of either) and a carbohydrateor glycomimetic. Such compounds may be combined with a pharmaceuticallyacceptable carrier or diluent to form a pharmaceutical composition. Thecompounds or compositions may be used in a method to modulate (e.g.,inhibit or enhance) a selectin-mediated function, such as inhibiting aselectin-mediated intercellular adhesion.

In one aspect of the present invention, compounds are provided thatcontain at least two components: (1) a BASA and (2) a carbohydrate orglycomimetic (or glycoconjugate of either). Examples of a BASA are setforth below. Preferred are the BASAs shown in FIGS. 1A-1H. Examples of acarbohydrate or glycomimetic are set forth below, and include sialylLe^(x), sialyl Le^(a) and glycomimetics of either. Preferred are thecarbohydrates or glycomimetics shown in FIG. 1l. One compound of thepresent invention is a combination of a BASA and a carbohydrate orglycomimetic, to yield a compound that modulates (e.g., inhibits orenhances) a selectin-mediated function. An example of aselectin-mediated function is a selectin-mediated intercellularadhesion. Preferred compounds are compounds 60, 61, 62 and 65 as shownin FIGS. 4, 5, 6 and 7, respectively. A compound of the presentinvention includes physiologically acceptable salts thereof. A compoundof the present invention in combination with a pharmaceuticallyacceptable carrier or diluent provides one composition of the presentinvention.

In another aspect of the present invention, methods are provided forusing a compound or composition of the present invention to modulate aselectin-mediated function. Such a compound or composition can be used,for example, to inhibit or enhance a selectin-mediated function, such asselectin-mediated intercellular interactions. A compound or compositioncan be used in a method to contact a cell expressing a selectin in anamount effective to modulate the selectin's function. A compound orcomposition can be used in a method to administer to a patient, who isin need of having inhibited the development of a condition associatedwith an excessive selectin-mediated function (such as an excessiveselectin-mediated intercellular adhesion), in an amount effective toinhibit the development of such a condition. Examples of such conditionsinclude inflammatory diseases, autoimmune diseases, infection, cancer,shock, thrombosis, wounds, burns, reperfusion injury, platelet-mediateddiseases, leukocyte-mediated lung injury, spinal cord damage, digestivetract mucous membrane disorders, osteoporosis, arthritis, asthma andallergic reactions. A compound or composition can be used in a method toadminister to a patient who is the recipient of a transplanted tissue inan amount effective to inhibit rejection of the transplanted tissue. Acompound or composition can be used in a method in an amount effectiveto target an agent to a selectin-expressing cell by contacting such acell with the agent linked to the compound or composition. A compound orcomposition can be used in the manufacture of a medicament, for examplefor any of the uses recited above.

In another aspect of the present invention, compounds are provided (andpharmaceutical compositions thereof, uses of such compounds orcompositions in the uses set forth above, and in the manufacture ofmedicaments) having one or more of the following structures:

(a)

wherein n=0 or 1;

X¹=—PO₂M, —SO₂M, or —CF₃;

R¹=—OH or —CO₂R⁴, wherein R⁴=—H or —(CH₂)_(m)—CH₃, wherein m=0-3;

R²=—H, —PO₃M₂, —SO₃M₂, —CH₂—PO₃M₂, —CH₂—SO₃M₂ or —CF₃

R⁵ and R⁶ are independently selected from —H, —CO₂—R⁷ and —NH—R⁸,wherein R⁷ and R⁸ are independently selected from hydrogen and moietiescomprising one or more of an alkyl group, an aromatic moiety, an aminogroup or a carboxy group;

R⁹ and R¹⁰ are independently selected from —H, —(CH₂)_(m)—CH₃; —CH₂—Ar,—CO—Ar, wherein m=0-3 and Ar=an aromatic moiety; and

M is selected from hydrogen, sodium, potassium and otherpharmaceutically acceptable counterions; or

wherein R₁ and R₂ are independently selected from the group consistingof (i) hydrogen, (ii) moieties comprising one or more of an alkyl group,an aromatic moiety, an amino group or a carboxy group, and (iii) —CO—R₃,wherein R₃ comprises an aromatic moiety; and

M is selected from hydrogen, sodium, potassium and otherpharmaceutically acceptable counterions.

These and other aspects of the present invention will become apparentupon reference to the following detailed description and attacheddrawings. All references disclosed herein are hereby incorporated byreference in their entirety as if each was incorporated individually.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

FIGS. 1A-1H show structures of representative BASA components of theselectin modulators as described herein. The compounds illustrated inthese figures include BASA portions and analogues. FIG. 1l showsstructures of representative carbohydrate or glycomimetic components ofthe selectin modulators as described herein.

FIG. 2 is a diagram illustrating the synthesis of a representative BASA.

FIG. 3 is a diagram illustrating the synthesis of a representative BASA.

FIG. 4 is a diagram illustrating the synthesis of a BASA analogue linkedto a carbohydrate moiety to form a selectin modulator of the presentinvention.

FIG. 5 is a diagram illustrating the synthesis of a BASA analogue linkedto a carbohydrate moiety to form a selectin modulator of the presentinvention.

FIG. 6 is a diagram illustrating the synthesis of a BASA analogue linkedto a carbohydrate moiety to form a selectin modulator of the presentinvention.

FIG. 7 is a diagram illustrating the synthesis of a BASA analogue linkedto a glycomimetic to form a selectin modulator of the present invention.

FIG. 8 is a bar graph illustrating the ability of BASA analogues toinhibit P-selectin function. Inhibition is shown as the amount of BASAanalogue required to inhibit P-selectin binding in an ELISA assay by 50%(IC₅₀).

FIG. 9 is a table illustrating the ability of BASA analogues to inhibitP-selectin function in ELISA and cell-based assays.

FIG. 10 is a table illustrating the inhibition of P-selectin-mediatedcell adhesion under flow by a BASA analogue linked to Sialyl Le^(a)(compound 62).

FIG. 11 is a bar graph illustrating the ability of a BASA analoguelinked to a glycomimetic of Sialyl Le^(x) (compound 65) to inhibitthioglycollate-induced peritonitis in a mouse model.

DETAILED DESCRIPTION OF THE INVENTION

As noted above, the present invention provides, in one aspect, selectinmodulators, compositions thereof and methods for modulatingselectin-mediated functions. Such modulators may be used in vitro or invivo, to modulate (e.g., inhibit or enhance) selectin-mediated functionsin a variety of contexts, discussed in further detail below. Examples ofselectin-mediated functions include intercellular adhesion and theformation of new capillaries during angiogenesis.

SELECTIN MODULATORS

The term “selectin modulator,” as used herein, refers to a molecule(s)that modulates (e.g., inhibits or enhances) a selectin-mediatedfunction, such as selectin-mediated intercellular interactions, and thatcomprises at least one of the following BASA:

(a) a BASA (or a salt thereof);

(b) a portion of a BASA that retains the ability to modulate (e.g.,inhibit or enhance) a selectin-mediated function; or

(c) an analogue of a BASA, or an analogue of a portion of a BASA, thathas the ability to modulate (e.g., inhibit or enhance) aselectin-mediated function;

wherein at least one of (a), (b) or (c) is linked to one or moreselectin binding carbohydrate or glycomimetic (or glycoconjugates ofeither).

A selectin modulator may consist entirely of one or more of the aboveBASA elements linked to one or more carbohydrate or glycomimetic, or maycomprise one or more additional molecular components. Within certainpreferred embodiments, as described in more detail below, a selectinmodulator comprises one of the above BASA elements linked to acarbohydrate moiety, such as sialyl-Lewis^(x) (SLe^(x)) orsialyl-Lewis^(a) (SLe^(a)), or linked to a glycomimetic, such as aglycomimetic of SLe^(x) or SLe^(a). The selectin modulators of thepresent invention are, surprisingly, significantly more potent than theindividual components alone or additively.

Within the present invention, BASAs are low molecular weight sulfatedcompounds which have the ability to interact with a selectin. Theinteraction modulates or assists in the modulation (e.g., inhibition orenhancement) of a selectin-mediated function (e.g., an intercellularinteraction). They exist as either their protonated acid form, or as asodium salt, although sodium may be replaced with potassium or any otherpharmaceutically acceptable counterion. A representative BASA has thefollowing structure:

Portions of BASA that retain the ability to interact with a selectin(which interaction modulates or assists in the modulation of aselectin-mediated function as described herein) are also selectinmodulators within the context of the present invention. Such portionsgenerally comprise at least one aromatic ring present within the BASAstructure. Within certain embodiments, a portion may comprise a singlearomatic ring, multiple such rings or half of a symmetrical BASAmolecule.

As noted above, analogues of BASA and portions thereof (both of whichpossess the biological characteristic set forth above) are alsoencompassed, e.g., by the BASA component of the selectin modulators,within the present invention. As used herein, an “analogue” is acompound that differs from BASA or a portion thereof because of one ormore additions, deletions and/or substitutions of chemical moieties,such that the ability of the analogue to inhibit a selectin-mediatedinteraction is not diminished. For example, an analogue may contain S toP substitutions (e.g., a sulfate group replaced with a phosphate group).Other possible modifications include: (a) modifications to ring size(e.g., any ring may contain between 4 and 7 carbon atoms); (b)variations in the number of fused rings (e.g., a single ring may bereplaced with a polycyclic moiety containing up to three fused rings, apolycyclic moiety may be replaced with a single unfused ring or thenumber of fused rings within a polycyclic moiety may be altered); (c)ring substitutions in which hydrogen atoms or other moieties covalentlybonded to a carbon atom within an aromatic ring may be replaced with anyof a variety of moieties, such as F, Cl, Br, I, OH, O-alkyl (C1-8), SH,NO₂, CN, NH₂, NH-alkyl (C1-8), N-(alkyl)₂, SO₃M (where M=H⁺, Na⁺, K⁺ orother pharmaceutically acceptable counterion), CO₂M, PO₄M₂, SO₂NH₂,alkyl (C1-8), aryl (C6-10), CO₂-alkyl (C1-8), —CF₂X (where X can be H,F, alkyl, aryl or acyl groups) and carbohydrates; and (d) modificationsto linking moieties (i.e., moieties located between rings in the BASAmolecule) in which groups such as alkyl, ester, amide, anhydride andcarbamate groups may be substituted for one another.

Certain BASA portions and analogues contain one of the following genericstructures:

Within this structure, n may be 0 or 1, X¹ may be —PO₂M, —SO₂M or —CF₂—(where M is a pharmaceutically acceptable counterion such as hydrogen,sodium or potassium), R¹ may be —OH, —F or —CO₂R⁴ (where R⁴ may be —H or—(CH₂)_(m)—CH₃ and m is a number ranging from 0 to 3, R² may be —H,—PO₃M₂, —SO₃M₂, —CH₂—PO₃M₂, —CH₂—SO₃M₂, —CF₃ or —(CH₂)_(m)—C(R⁶)H—R⁵ orR⁹—N(R¹⁰)—, R³ may be —H, —(CH₂)_(m)—C(R⁶)H—R⁵ or R⁹—N(R¹⁰)— (where R⁵and R⁶ may be independently selected from —H, —CO₂—R⁷ and —NH—R⁸, R⁷ andR⁸ may be independently selected from hydrogen and moieties comprisingone or more of an alkyl group, an aromatic moiety, an amino group or acarboxy group, and R⁹ and R¹⁰ may be independently selected from —H,—(CH₂)_(m)—CH₃; —CH₂—Ar, —CO—Ar, where m is a number ranging from 0 to 3and Ar is an aromatic moiety (i.e., any moiety that comprises at leastone substituted or unsubstituted aromatic ring, wherein the ring isdirectly bonded to the —CH₂— or —CO— group indicated above)).

Other portions and analogues of BASA comprise the generic structure:

Within this structure, R₁ and R₂ may be independently selected from (i)hydrogen, (ii) moieties comprising one or more of an alkyl group, anaromatic moiety, an amino group or a carboxy group, and (iii) —CO—R₃(where R₃ comprises an alkyl or aromatic moiety as described above) andM is a pharmaceutically acceptable counterion.

The individual compounds, or groups of compounds, derived from thevarious combinations of the structures and substituents describedherein, are disclosed by the present application to the same extent asif each compound or group of compounds was set forth individually. Thus,selection of particular structures and/or particular substituents iswithin the scope of the present invention.

Representative BASA portions and analogues are included in the compoundsshown in FIGS. 1A-1H. It will be apparent to those of ordinary skill inthe art that modifications may be made to the compounds shown withinthese figures, without adversely affecting the ability to function asselectin modulators. Such modifications include deletions, additions andsubstitutions as described above.

Certain selectin modulator components are commercially available from,for example, Sigma-Aldrich, Toronto Research Chemicals, Calbiochem andothers. Others may be prepared using well known chemical synthetictechniques from available compounds. General synthetic methods for thesynthesis of selectin modulators include the following: Amide formationof a primary or secondary amine or aniline can be accomplished viareaction with an acyl halide or carboxylic acid (see FIGS. 2 and 3).N-linked alkyl compounds are prepared by reductive amination of theamine/aniline with an aldehyde followed by imine reduction via sodiumcyanoborohydride (see FIG. 6). Biphenyl compounds are easily prepared byreaction of suitable aryl bromide/iodides with appropriate boronic acidsvia Suzuki/Negishi conditions (see FIG. 2). Reduction of nitro groupscan be selectively accomplished in the presence of other sensitivesubstrates by palladium catalyzed hydrogenation (see FIGS. 2 and 3).

A BASA component (such as those set forth above) is linked (e.g.,covalently attached with or without a spacer group) to one or moreselectin binding carbohydrate moieties or glycomimetics (orglycoconjugates of either) to form a selectin modulator of the presentinvention. The carbohydrate moieties selected include sialyl-Lewis^(x)(SLe^(x)), sialyl-Lewis^(a) (SLe^(a)), and glycomimetics andglycoconjugates of these and related carbohydrates. Carbohydratemoieties comprising sialic acid-containing glycomimetics, which may beincluded in a selectin modulator, are found in U.S. Pat. Nos.6,187,754B1 and 6,169,077B1 (for some structures see compounds 57 and 59in FIG. 1l). These include glycomimetics of SLe^(x) and SLe^(a) withincreased binding affinity for the selectins over the parent compounds.Non-sialic acid containing glycomimetics are illustrated by thecompounds provided in the table on page 2897 of Helvetica Chemica ActaVol. 83 (2000) and by Table 1 on page 3646 of Angew. Chem. Int. Ed. Vol.40, No. 19 (2001). These glycomimetics include the replacement of sialicacid with cyclohexyllactic acid as well as additional functional groupchanges introduced on additional glycans (for some structures seecompounds 55, 56 and 58 in FIG. 1l).

The attachment of a BASA to a carbohydrate moiety can be accomplished ina variety of ways to form a selectin modulator. The simplestcarbohydrate moiety attachment method is reductive amination of the BASAto the carbohydrate moiety's reducing end (the anomerichydroxyl/aldehyde-see FIG. 6). This is accomplished by simple reactionof the BASA to the reducing carbohydrate moiety and subsequent reductionof the imine formed. The loss of the cyclic nature of the sugar reactedwith, limits the usefulness of this method. The most general approachentails the simple attachment of an activated linker to the carbohydratemoiety via an O, S or N heteroatom (or C atom for C-linked glycosides)at the anomeric position of the glycan. The methodology of suchattachments has been extensively researched and anomeric selectivity iseasily accomplished by proper selection of methodology and/or protectinggroups. Examples of potential glycosidic synthetic methods include Lewisacid catalyzed bond formation with halogen or peracetylated sugars(Koenigs Knorr), trichloroacetamidate bond formation, thioglycosideactivation and coupling, glucal activation and coupling, n-pentenylcoupling, phosphonate ester homologation (Horner-Wadsworth-Emmonsreaction), and many others. Alternatively, linkers could be attached topositions on the carbohydrate moieties other than the anomeric. The mostaccessible site for attachment is at the six hydroxyl (6-OH) position ofthe sugar/sugar mimetics (a primary alcohol). The attachment of a linkerat the 6-OH can be easily achieved by a variety of means. Examplesinclude reaction of the oxy-anion (alcohol anion formed by deprotonationwith base) with an appropriate electrophile such as an alkyl/acylbromide, chloride or sulfonate ester, activation of the alcohol viareaction with a sulfonate ester chloride or POCl₃ and displacement witha subsequent nucleophile, oxidation of the alcohol to the aldehyde orcarboxylic acid for coupling, or even use of the Mitsunobu reaction tointroduce differing functionalities. Once attached the carbohydratelinker is then functionalized for reaction with a suitable nucleophileon the selectin modulator (or vice versa). This is often accomplished byuse of thiophosgene and amines to make thiourea-linkedheterobifunctional ligands (see FIG. 4), diethyl squarate attachment(again with amines—see FIG. 5) and/or simple alkyl/acylation reactions.Additional methods that could be utilized include FMOC solid or solutionphase synthetic techniques amenable for carbohydrate and peptidecoupling (for novel glycopeptides and glycopeptidomimetics) andchemo-enzymatic synthesis techniques possibly utilizing glycosyl/fucosyltransferases and/or oligosaccharyltransferase (OST).

Although selectin modulators as described herein may sufficiently targeta desired site in vivo, it may be beneficial for certain applications toinclude an additional targeting moiety to facilitate targeting to one ormore specific tissues. As used herein, a “targeting moiety,” may be anysubstance (such as a compound or cell) that, when linked to a modulatingagent enhances the transport of the modulator to a target tissue,thereby increasing the local concentration of the modulator. Targetingmoieties include antibodies or fragments thereof, receptors, ligands andother molecules that bind to cells of, or in the vicinity of, the targettissue. Linkage is generally covalent and may be achieved by, forexample, direct condensation or other reactions, or by way of bi- ormulti-functional linkers.

For certain embodiments, it may be beneficial to also, or alternatively,link a drug to a selectin modulator. As used herein, the term “drug”refers to any bioactive agent intended for administration to a mammal toprevent or treat a disease or other undesirable condition. Drugs includehormones, growth factors, proteins, peptides and other compounds.Examples of potential drugs include antineoplastic agents (such as5-fluorouracil and distamycin), integrin agonist/antagonists (such ascyclic-RGD peptide), cytokine agonist/antagonists, histamineagonist/antagonists (such as diphenhydramine and chlorpheniramine),antibiotics (such as aminoglycosides and cephalosporins) and redoxactive biological agents (such as glutathione and thioredoxin). In otherembodiments, diagnostic or therapeutic radionuclides may be linked to aselectin modulator. In many embodiments, the agent may be linkeddirectly or indirectly to a selectin modulator.

EVALUATING INHIBITION OF SELECTIN-MEDIATED INTERCELLULAR ADHESION

Modulating agents as described above are capable, for example, ofinhibiting selectin-mediated cell adhesion. This ability may generallybe evaluated using any of a variety of in vitro assays designed tomeasure the effect on adhesion between selectin-expressing cells (e.g.,adhesion between leukocytes and platelets or endothelial cells). Forexample, such cells may be plated under standard conditions that, in theabsence of modulator, permit cell adhesion. In general, a modulator isan inhibitor of selectin-mediated cell adhesion if contact of the testcells with the modulator results in a discernible disruption of celladhesion. For example, in the presence of modulators (e.g., micromolarlevels), disruption of adhesion between leukocytes and platelets and/orendothelial cells may be determined visually within approximatelyseveral minutes, by observing the reduction of cells interacting withone another.

SELECTIN MODULATOR FORMULATIONS

Modulators as described herein may be present within a pharmaceuticalcomposition. A pharmaceutical composition comprises one or moremodulators in combination with one or more pharmaceutically orphysiologically acceptable carriers, diluents or excipients. Suchcompositions may comprise buffers (e.g., neutral buffered saline orphosphate buffered saline), carbohydrates (e.g., glucose, mannose,sucrose or dextrans), mannitol, proteins, polypeptides or amino acidssuch as glycine, antioxidants, chelating agents such as EDTA orglutathione, adjuvants (e.g., aluminum hydroxide) and/or preservatives.Within yet other embodiments, compositions of the present invention maybe formulated as a lyophilizate. Compositions of the present inventionmay be formulated for any appropriate manner of administration,including for example, topical, oral, nasal, intravenous, intracranial,intraperitoneal, subcutaneous, or intramuscular administration.

A pharmaceutical composition may also, or alternatively, contain one ormore active agents, such as drugs (e.g., those set forth above), whichmay be linked to a modulator or may be free within the composition.

The compositions described herein may be administered as part of asustained release formulation (i.e., a formulation such as a capsule orsponge that effects a slow release of modulating agent followingadministration). Such formulations may generally be prepared using wellknown technology and administered by, for example, oral, rectal orsubcutaneous implantation, or by implantation at the desired targetsite. Carriers for use within such formulations are biocompatible, andmay also be biodegradable; preferably the formulation provides arelatively constant level of modulating agent release. The amount ofmodulating agent contained within a sustained release formulationdepends upon the site of implantation, the rate and expected duration ofrelease and the nature of the condition to be treated or prevented.

Selectin modulators are generally present within a pharmaceuticalcomposition in a therapeutically effective amount. A therapeuticallyeffective amount is an amount that results in a discernible patientbenefit, such as increased healing of a condition associated with excessselectin-mediated function (e.g., intercellular adhesion), as describedbelow.

SELECTIN MODULATOR METHODS OF USE

In general, the modulating agents and compositions described herein maybe used for enhancing or inhibiting a selectin-mediated function. Suchenhancement or inhibition may be achieved in vitro and/or in vivo in awarm-blooded animal, preferably in a mammal such as a human, providedthat a selectin-expressing cell is ultimately contacted with amodulator, in an amount and for a time sufficient to enhance or inhibitselectin-mediated function.

Within certain aspects, the present invention provides methods forinhibiting the development of a condition associated with aselectin-mediated function, such as intercellular adhesion. In general,such methods may be used to prevent, delay or treat such a condition. Inother words, therapeutic methods provided herein may be used to treat adisease, or may be used to prevent or delay the onset of such a diseasein a patient who is free of disease or who is afflicted with a diseasethat is not associated with a selectin-mediated function.

A variety of conditions are associated with a selectin-mediatedfunction. Such conditions include, for example, tissue transplantrejection, platelet-mediated diseases (e.g., atherosclerosis andclotting), hyperactive coronary circulation, acute leukocyte-mediatedlung injury (e.g., adult respiratory distress syndrome (ARDS)), Crohn'sdisease, inflammatory diseases (e.g., inflammatory bowel disease),autoimmune diseases (MS, myasthenia gravis), infection, cancer (andmetastasis), thrombosis, wounds (and wound-associated sepsis), burns,spinal cord damage, digestive tract mucous membrane disorders(gastritis, ulcers), osteoporosis, rheumatoid arthritis, osteoarthritis,asthma, allergy, psoriasis, septic shock, traumatic shock, stroke,nephritis, atopic dermatitis, frostbite injury, adult dyspnoea syndrome,ulcerative colitis, systemic lupus erythematosus, diabetes andreperfusion injury following ischaemic episodes. Selectin modulators mayalso be administered to a patient prior to heart surgery to enhancerecovery. Other uses include for pain management and for undesirableangiogenesis, e.g., associated with cancer.

Selectin modulators of the present invention may be administered in amanner appropriate to the disease to be treated (or prevented).Appropriate dosages and a suitable duration and frequency ofadministration may be determined by such factors as the condition of thepatient, the type and severity of the patient's disease and the methodof administration. In general, an appropriate dosage and treatmentregimen provides the modulating agent(s) in an amount sufficient toprovide therapeutic and/or prophylactic benefit. Within particularlypreferred embodiments of the invention, a selectin modulator may beadministered at a dosage ranging from 0.001 to 100 mg/kg body weight, ona regimen of single or multiple daily doses. Appropriate dosages maygenerally be determined using experimental models and/or clinicaltrials. In general, the use of the minimum dosage that is sufficient toprovide effective therapy is preferred. Patients may generally bemonitored for therapeutic effectiveness using assays suitable for thecondition being treated or prevented, which will be familiar to those ofordinary skill in the art.

Selectin modulators may also be used to target substances to cells thatexpress a selectin. Such substances include therapeutic agents anddiagnostic agents. Therapeutic agents may be a molecule, virus, viralcomponent, cell, cell component or any other substance that can bedemonstrated to modify the properties of a target cell so as to providea benefit for treating or preventing a disorder or regulating thephysiology of a patient. A therapeutic agent may also be a prodrug thatgenerates an agent having a biological activity in vivo. Molecules thatmay be therapeutic agents may be, for example, polypeptides, aminoacids, nucleic acids, polynucleotides, steroids, polysaccharides orinorganic compounds. Such molecules may function in any of a variety ofways, including as enzymes, enzyme inhibitors, hormones, receptors,antisense oligonucleotides, catalytic polynucleotides, anti-viralagents, anti-tumor agents, anti-bacterial agents, immunomodulatingagents and cytotoxic agents (e.g., radionuclides such as iodine,bromine, lead, palladium or copper). Diagnostic agents include imagingagents such as metals and radioactive agents (e.g., gallium, technetium,indium, strontium, iodine, barium, bromine and phosphorus-containingcompounds), contrast agents, dyes (e.g., fluorescent dyes andchromophores) and enzymes that catalyze a colorimetric or fluorometricreaction. In general, therapeutic and diagnostic agents may be attachedto a selectin modulator using a variety of techniques such as thosedescribed above. For targeting purposes, a selectin modulator may beadministered to a patient as described herein. Since selectins arechemotactic molecules for endothelial cells involved in the formation ofnew capillaries during angiogenesis, a selectin modulator may be used totarget a therapeutic agent for killing a tumor's vasculature. A selectinmodulator may also be used for gene targeting.

Selectin modulators may also be used in vitro, e.g., within a variety ofwell known cell culture and cell separation methods. For example,modulators may be linked to the interior surface of a tissue cultureplate or other cell culture support, for use in immobilizingselectin-expressing cells for screens, assays and growth in culture.Such linkage may be performed by any suitable technique, such as themethods described above, as well as other standard techniques.Modulators may also be used, for example, to facilitate cellidentification and sorting in vitro, permitting the selection of cellsexpressing a selectin (or different selectin levels). Preferably, themodulator(s) for use in such methods are linked to a detectable marker.Suitable markers are well known in the art and include radionuclides,luminescent groups, fluorescent groups, enzymes, dyes, constantimmunoglobulin domains and biotin. Within one preferred embodiment, amodulator linked to a fluorescent marker, such as fluorescein, iscontacted with the cells, which are then analyzed by fluorescenceactivated cell sorting (FACS).

Although less preferred, a BASA component (i.e., BASA, portion of aBASA, analogue of a BASA, or analogue of a portion of a BASA) of aselectin modulator may be prepared alone as a pharmaceuticalcomposition, and used in the methods above as a compound (orcomposition) independent of or in combination with the selectinmodulators of the present invention. The above description is applied toa BASA component.

All compounds of the present invention or useful thereto, includephysiologically acceptable salts thereof.

The following Examples are offered by way of illustration and not by wayof limitation.

EXAMPLES Example 1 Preparation of Representative Selectin Modulators

This Example illustrates the synthesis of representative selectinmodulators or components thereof; specifically, compounds 39, 22, 60,61, 62, and 65 (FIGS. 2 thru 7 respectively).

SYNTHESIS OF 39 (FIG. 2):

Suzuki COUPLING

4-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-benzoic acid (0.004mol, 1 eq) and KOAc (0.012 mol, 3 eq) are placed in THF (25 ml) creatinga slurry.

PdCl₂(dppf) (0.00012 mol, 3 mol %) and p-bromo-nitrobenzene (0.005 mol,1.2 eq) are then added to the solution with stirring and the solution isheated gently to 80° C. After 6 hrs the reaction is complete by TLC(20:1 CH₂Cl₂/CH₃OH). The reaction mixture is evaporated to dryness,dissolved in CH₂Cl₂ (30 ml) and washed with distilled water andsaturated NaHCO₃. The resultant biphenyl compound is taken directly tothe next step.

CARBODIIMIDE COUPLING

4′-Nitro-biphenyl-4-carboxylic acid (0.004 mol, 1 eq), dimethyl aminopyridine (1 crystal, cat.) and EDCl (0.0041 mol, 1.05 eq) are dissolvedin DMF (or THF, 20 ml) and allowed to react at room temperature for 10min. 8-Amino-naphthalene-1,3,5-trisulfonic acid is added to the reactionmixture with stirring and the reaction is allowed to proceed at roomtemperature under nitrogen for 48 hrs. The reaction mixture is thenevaporated to dryness and purified by reverse phase chromatography (C18column, 80/20 CH₃CN/H₂O-1% TFA to 50/50 CH₃CN/H₂O).

HYDROGENATION

8-[(4′-Nitro-biphenyl-4-carbonyl)-amino]-naphthalene-1,3,5-trisulfonicacid (1 eq) and 10% Pd (10 mol %) on carbon are placed in EtOAc (orCH₃OH). The solution is degassed and an atmosphere of H₂ is generatedwithin the reaction vessel. The reaction is allowed to proceed until theuptake of H₂ ceases and TLC indicates the disappearance of startingmaterial (˜12 hrs). The palladium precipitate is removed by filtrationthrough a bed of celite and the filtrate is evaporated to dryness givingcompound 39.

SYNTHESIS OF 22 (FIG. 3):

ACID CHLORIDE COUPLING

8-Amino-naphthalene-1,3,5-trisulfonic acid (0.004 mol, 1 eq) anddiisopropyl ethyl amine (6 eq) are placed in DMF (20 ml) and cooled to0° C. 3-nitro-4-methyl benzoyl chloride (0.005 mol, 1.2 eq) is dissolvedin DMF and added dropwise to the cooled solution over 10 min. Thereaction is allowed to proceed at 0° C. for 3 hrs. The reaction mixtureis washed with 0.1M HCl (25 ml), frozen and evaporated to dryness. Theresultant syrup is used without purification in the next step.

HYDROGENATION

8-(4-Methyl-3-nitro-benzoylamino)-naphthalene-1,3,5-trisulfonic acid (1eq) and 10% Pd on carbon (10 mol %) are placed in CH₃OH. The solution isdegassed and an atmosphere of H₂ is generated within the reactionvessel. The reaction is allowed to proceed until the uptake of H₂ ceasesand TLC indicates the disappearance of starting material (12 hrs). Thepalladium precipitate is removed by filtration through a bed of celiteand the filtrate is evaporated to dryness giving the reduced compound8-(3-Amino-4-methyl-benzoylamino)-naphthalene-1,3,5-trisulfonic acid.

ACID CHLORIDE COUPLING

8-(3-Amino-4-methyl-benzoylamino)-naphthalene-1,3,5-trisulfonic acid(0.004 mol, 1 eq) and diisopropyl ethyl amine (6 eq) are placed in DMF(15 ml) and cooled to 0° C. 3-Nitro-benzoyl chloride (0.005 mol, 1.2 eq)is dissolved in DMF (5 ml) and added dropwise to the cooled solutionover 10 min. The reaction is allowed to proceed at 0° C. for 3 hrs. Thereaction mixture is washed with 0.1M HCl (25 ml) and evaporated todryness. The compound is purified by reverse phase chromatography (C18column, 80/20 CH₃CN/H₂O-1% TFA to 50/50 CH₃CN/H₂O).

SYNTHESIS OF 60 (FIG. 4)

ACTIVATED ESTER SYNTHESIS

The Lemieux ester of SLe^(x) (1 eq) is dissolved in H₂O and1,2-diaminoethane (3 eq) is added with stirring. The solution is heatedto 70° C. for 50 hrs under nitrogen. The solution is evaporated todryness and the compound purified by reverse phase chromatography (C18column, 80/20 CH₃CN/H₂O-1% TFA to 50/50 CH₃CN/H₂O).

THIOISOCYANATE FORMATION

The Lemieux ester-amine (1 eq) is dissolved in H₂O and reacted withthiophosgene (3 eq-warning highly toxic) for 3 hrs. The solution is thenwashed with CH₂Cl₂ to remove unreacted thiophosgene and the aqueouslayer is collected and evaporated to dryness. The compound is used as isfor the next reaction.

FORMATION OF 60

The activated thioisocyanate (1 eq) is dissolved in an NH₄OAc buffersolution of pH 9 and8-(2-Amino-acetylamino)-naphthalene-1,3,5-trisulfonic acid (1.2 eq) isadded with stirring. The reaction is allowed to stir at room temperaturefor 6 hrs under nitrogen. The compound is then purified by reverse phasechromatography (C18 column, 80/20 CH₃CN/H₂O-1% TFA to 50/50 CH₃CN/H₂O).

SYNTHESIS OF 61 (FIG. 5)

ACTIVATED ESTER SYNTHESIS

The Lemieux ester of SLe^(x) (1 eq) is dissolved in H₂O and1,2-diaminoethane (3 eq) is added with stirring. The solution is heatedto 80° C. for 50 hrs under nitrogen. The solution is evaporated todryness and the compound purified by reverse phase chromatography (C18column, 80/20 CH₃CN/H₂O-1% TFA to 50/50 CH₃CN/H₂O).

SQUARATE ESTER FORMATION

The Lemieux ester-amine (1 eq) is dissolved in 0.1M Hepes buffer of pH 7and reacted with squaric acid diethyl ester (3 eq) for 3 hrs. Thesolution is then washed with CH₂Cl₂ and the aqueous layer is collectedand evaporated to dryness. The resultant powder is used as is in thenext step.

FORMATION OF 61

The activated squarate ester-sugar (1 eq) is dissolved in buffer of pH9.5 and 8-(3-Amino-benzoylamino)-naphthalene-1,3,5-trisulfonic acid (1.2eq) is added with stirring. The reaction is allowed to stir at roomtemperature for 6 hrs under nitrogen. The solution is evaporated todryness and the compound is purified by reverse phase chromatography(C18 column, 80/20 CH₃CN/H₂O-1% TFA to 50/50 CH₃CN/H₂O).

SYNTHESIS OF 62 (FIG. 6) REDUCTIVE AMINATION

SLe^(a) (20 μmol, 1 eq) is dissolved in acetic acid buffered H₂O of pH 4(100 μl) along with BASA 41 (20 μmol, 1 eq). The reaction is allowed toproceed at room temperature under nitrogen for 12 hrs. The resultantimine formed is then reduced by reaction with NaCNBH₃(30 μmol, 1.5 eq)in CH₃CH₂OH/H₂O (2:1). The solution is then evaporated to dryness andpurified by reverse phase chromatograpy (C18 column, 80/20 CH₃CN/H₂O-1%TFA to 50/50 CH₃CN/H₂O).

SYNTHESIS OF 65 (FIG. 7)

ACTIVATED ESTER SYNTHESIS

Compound 63 (1.8 μmol, 1 eq) is dissolved in 1,2-diaminoethane (50 μl,XS) with stirring. The solution is heated to 70° C. for 50 hrs undernitrogen. The solution is then evaporated to dryness and the compound ispurified by reverse phase chromatography (C18 column, 80/20 CH₃CN/H₂O-1%TFA to 50/50 CH₃CN/H₂O).

SQUARATE ESTER FORMATION

The 63-amine (5.40 μmol, 1 eq) is dissolved in 0.1M Hepes buffer of pH 7and reacted with squaric acid diethyl ester (80 μmol, XS) for 24 hrs.The solution is then washed with CH₂Cl₂ and the aqueous layer iscollected and evaporated to dryness. The resultant powder is purified bycolumn chromatography (Sephadex-G25, 100% H₂O).

FORMATION OF 65

The activated 63-squarate ester-sugar (5.40 μmol, 1 eq) is dissolved inNaHCO₃ buffer of pH 9.5 (2 ml) and BASA 64 (6.48 μmol , 1.2 eq) is addedwith stirring. The reaction is allowed to stir at room temperature for 3hrs under nitrogen. The solution is evaporated to dryness and compound65 is purified by column chromatography (Sephadex-G25, 100% H₂O).

Example 2 Inhibition of P-Selectin by BASE Analogues

This Example illustrates the ability of various BASA analogues toinhibit P-selectin function in ELISA and cell-based assays.

LIGAND BINDING ASSAY FOR P-SELECTIN

The neoglycoprotein SLea-HSA (IsoSep AB, Tullinge, Sweden) is incubatedin wells of a 96-well microtiter plate (Falcon probind) at 100 ng/wellin 50 mM Tris, 0.15M NaCl, 2 mMCaCl₂, pH 7.5 (TBS) overnight at 4° C.After the coating incubation, the wells are blocked with 100 μL/well of2% bovine serum albumin (BSA) at room temperature for 2 hours.

Test compounds are serially diluted in a second 96 well plate (Plate 2;U-bottomed, low binding plate) in Dulbecco's PBS, pH 7.0 (DPBS,Biofluids, Inc.). Plate 1 is washed five times with DBPS and thecontents of plate 2 are transferred to plate 1. An equal volume ofP-selectin/hlg chimera (GlycoTech Corp.) is added to the wells at 4μg/ml in 1% BSA, DPBS, pH 7.0. and the plate is incubated at roomtemperature for 2 hr. After incubation, the plate is washed five timesand 100 μl/well of 1 μg/ml of peroxidase-labeled goat anti-human Ig (KPLlabs, Gaithersburg, Md.) is added to plate 1. After incubation at roomtemperature for 1 hour, the plate is washed five times and TMB substrate(KPL labs, Gaithersburg, Md.) is added to each well. After five minutesthe reaction is stopped by adding 100 μl/well of 1M H₃PO₄ and theabsorbance of light at 450 nm is read by a microtiter plate reader.

HIGHLY SENSITIVE LIGAND BINDING ASSAY FOR P-SELECTIN

P-selectin/hlg chimera (GlycoTech Corp., Rockville, Md.) in 50 mM Tris,0.15M NaCl, 2 mM CaCl₂, pH 7.5 (TBS) is incubated in a microtiter plate(Plate 1, Falcon probind) for 2 hours at 37° C. After incubation, theplate is washed 5 times and 100 μl/well of 1% BSA in TBS is added toeach well and the plate is incubated at room temperature for 1 hr.

Test compounds are serially diluted in a second round bottomed lowbinding plate (plate 2). An equal volume of the conjugateSLexPAAbiotin-streptavidinHRP is added to each well. TheSLexPAAbiotin-streptavidinHRP is premade by mixing SLexPAA-biotin(GlycoTech Corp., Rockville, Md.) with streptavidinHRP (Sigma ChemicalCo., St Louis, Mo.).

Plate 1 is washed 5 times and the contents of Plate 2 are transferred toPlate 1. After incubation for 2 hours, the plate is washed 5 times and100 μl/well of TMB substrate (KPL labs, Gaithersburg, Md.) is added.After 10 minutes the reaction is stopped by adding 100 μl/well of 1MH₃PO₄ and the absorbance of light at 450 nm is read by a microtiterplate reader.

CELL-BASED ASSAY

Human umbilical vein endothelial cells (huvecs) are isolated fromumbilical cords. When the huvecs reach confluence in the T-175 flasks,they are passaged to 35mm tissue culture dishes coated with fibronectin(FN) (Gibco 33016-023). The dishes are used in 3-5 days once a confluentmonolayer is obtained.

Neutrophils are isolated from fresh blood the day of each experiment andused within five hours of the isolation. The isolated PMNs are suspendedin HBSS with Ca and Mg (Sigma H9269) and 12 mM Hepes (Biofluids, MD#305) at 10⁷ cells/ml for use in the flow assay.

A parallel plate flow chamber is used to study the rolling behaviorwhich is characteristic of cells in contact with selectins in thepresence of hydrodynamic flow. The parallel plate flow chamber(GlycoTech, Rockville, Md.) with a silicon rubber gasket is of acircular design to accommodate 35 mm tissue culture dishes (Corning)held in place by vacuum. The wall shear stress (τ_(w), dynes/cm²) isgiven by τ=6 μQ/a²b, where μ is the apparent viscosity of the media (forH₂O@37° C.=0.0076P), a is the channel height (i.e., gasket thickness of254 μm), b is the channel width (i.e., gasket width of 0.25 cm), and Qis the volumetric flow rate (ml/min).

Prior to the flow assay, the confluent huvec monolayers are incubatedwith IL-4. P-selectin antagonists and histamine are added to the PMNsprior to flow. The cell suspension of PMNs (10⁶ cells/ml) containing theantagonists is perfused through the chamber at a shear ratecorresponding to a wall shear stress of 0.9 dynes/cm². The cellsuspension is allowed to flow through the chamber for three minutesbefore digital images are collected.

The digital image system consists of a Silicon Graphics Indigo2workstation interfacing to Inovisionis IC300 digital image system. ThePMNs interacting with the huvecs are visualized using a Zeiss invertedstage microscope (ICM 405) operated in the phase contrast mode using a10× objective. A CCD camera is mounted on the microscope to provide thesignal to the digital image system. The experiments are recorded on avideo recorder. After three minutes of perfusing cells through the flowchamber, digital images are acquired at 7-10 different locations on eachof three dishes for every experimental condition.

The number of interacting cells (NIC) and the number of arresting cells(NAC) are determined by a segmentation program based on pixel intensityand size. Quantification of the rolling behavior is performed byanalysis of images containing the rolling cells as vertical streaks. Themeasure of rolling is the rolling index (RI) defined as the total area(i.e., total pixel count) of all the vertical streaks in each image.Results from the flow assay are summarized in FIG. 9. The antagonistsare found to be active in the low μM range. These data confirm theability of BASA analogues to inhibit P-selectin function.

Example 3 Thioglycollate-Induced Peritonitis in the Mouse

Peritonitis is induced in the mouse by intraperitoneal (i.p.) injectionof thioglycollate (time 0, t=0). Peritonitis was allowed to develop for4 hours. Test compounds were administered by intraperitoneal injected att=o. Antibody to

P-selectin was used as a positive control and sterile saline was used asa negative control. After 4 hours cells were removed from the peritonealcavity by a syringe and the number of neutrophils determined. Inhibitionof thioglycollate-induced peritonitis in the mouse by theheterobifunctional compound (cmpd 65) containing both glycomimetic (cmpd63) and BASA analogue (cmpd 64) is far better than either compoundalone.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention.

1. A compound or physiologically acceptable salt thereof, comprising a benzyl amino sulfonic acid (BASA) linked to a glycomimetic, wherein the glycomimetic binds a selectin.
 2. The compound or physiologically acceptable salt thereof according to claim 1 wherein the glycomimetic is a glycomimetic of sialyl Le^(x) or sialyl Le^(a).
 3. The compound according to claim 1 in combination with a pharmaceutically acceptable carrier or diluent.
 4. A compound or physiologically acceptable salt thereof comprising a compound according to claim 1 or claim 2 attached to a diagnostic or therapeutic agent.
 5. The compound according to claim 4 in combination with a pharmaceutically acceptable carrier or diluent. 6.-8. (canceled)
 9. A method of targeting an agent to a selectin-expressing cell, comprising contacting a cell expressing a selectin with a compound according to claim 4, or claim 5, in an amount effective to target a diagnostic or therapeutic agent to the cell.
 10. The compound according to claim 2 in combination with a pharmaceutically acceptable carrier or diluent. 